1. Field of the Invention
This invention relates to a general method for purification of polypeptides of moderate hydrophobicity to substantial homogeneity from a mixture such as a cell culture fluid in a single step.
2. Description of Background and Related Art
The large-scale, economic purification of proteins is increasingly an important problem for the biotechnology industry. Generally, proteins are produced by cell culture, using either mammalian or bacterial cell lines engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for that protein. Since the cell lines used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, and growth factors, usually supplied from preparations of animal serum. Separation of the desired protein from the mixture of compounds fed to the cells and from the by-products of the cells themselves to a purity sufficient for use as a human therapeutic poses a formidable challenge. Usually, the separation procedure is multi-step, requiring expensive apparatus and chromatography media. For review articles on this subject, see Ogez et al., Biotech. Adv., 7: 467-488 (1989) and Sofer, Bio/Technology, 4: 712-715 (1986).
Procedures for purification of proteins from cell debris initially depend on the site of expression of the protein. Some proteins can be caused to be secreted directly from the cell into the surrounding growth media; others are made intracellularly. For the latter proteins, the first step of a purification process involves lysis of the cell, which can be done by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments. Such disruption releases the entire contents of the cell into the homogenate, and in addition produces subcellular fragments that are difficult to remove due to their small size. These are generally removed by differential centrifugation or by filtration. The same problem arises, although on a smaller scale, with directly secreted proteins due to the natural death of cells in the course of the protein production run.
Once a clarified solution containing the protein of interest has been obtained, its separation from the other proteins produced by the cell is usually attempted using a combination of different chromatography techniques. These techniques separate mixtures of proteins on the basis of their charge, degree of hydrophobicity, or size. Several different chromatography resins are available for each of these techniques, allowing accurate tailoring of the purification scheme to the particular protein involved. Affinity chromatography, which exploits a specific interaction between the protein to be purified and a second protein (such as a specific antibody), may also be an option for some proteins.
The essence of each of these separation methods is that proteins can be caused either to move at different rates down a long column, achieving a physical separation that increases as they pass further down the column, or to adhere selectively to the separation medium, being then differentially eluted by different solvents. In some cases, the desired protein is separated from impurities when the impurities specifically adhere to the column, and the protein of interest does not, that is, the protein of interest is present in the "flow-through."
For some of these purification techniques, high-pressure liquid chromatography (HPLC) can be performed at preparative scale, allowing a high degree of separation using resins that would require prohibitive column sizes and extremely slow separation steps under more conventional conditions. However, this technique also requires expensive equipment and the use of chromatography columns and packings having the chemical and mechanical stability to permit hundreds or thousands of repeated purifications under high pressure.
Adsorption chromatography has been frequently used in the purification of small molecules such as steroids, and is now finding increasing use for large-scale protein purification. The ability of silica particles to adsorb protein with high capacity and affinity has been known for a long time. The major advance has been the determination of methods for desorption of proteins in high yield and in an active form.
In earlier applications of the method, elution was carried out with pH shifts [Edy et al., J. Gen. Virol., 33: 517-521 (1976)] or chaotropic salts. Whitman et al., J. Interferon Res., 1: 305-312 (1981). Chadha and Sulkowski, J. Interferon Res., 2: 229-234 (1982) introduced alkyl amines and organic solvents as eluants and achieved high recoveries of interferon. They have proposed a theory of mechanisms involved in the binding and desorption of proteins on glass. Chadha and Sulkowski, Prep. Biochem., 11: 467-482 (1981). The theory takes into account both the polar and non-polar forces involved in bonding proteins to silica, and describes the properties that a good eluant must possess to break both types of bonds.
Although many of the experiments have been carried out with "controlled-pore" glass, other porous glasses such as ordinary silica particles can give equivalent performance at a fraction of the cost. Bare silica, which is underivatized silica particles, is primarily sold as a desiccation agent. Silica is a popular backbone for the production of chromatography media, since it is inexpensive and is sufficiently chemically active to accept the addition of a wide range of substituent groups. See Anspach et al., J. Chromatog., 443: 45-54 (1988), where various size-exclusion chromatography columns were examined in eluting protein, and ionic strength of the eluent was changed by varying the salt (sodium chloride) concentration of the phosphate buffer, so as to affect the elution volume of proteins differently for two different columns. In addition, silica particles are robust, allowing repeated use of a column of derivatized silica for purification, and give good solvent flow through the column. Underivatized silica is not generally used as a chromatography medium, although it is used as a filter to remove cellular debris and highly hydrophobic impurities.
Silica particles are available in a variety of forms, with different sizes of particle and pore size within the particle. The size of particle chiefly determines the packing properties of the material, which determine the rate of flow and the back pressure when the material is used as a column. The pore size, however, determines the size of protein that has access to the interior of the pore. Kiselev et al., in Column Chromatography, 5th Int. Symposium (Lausanne, 7-10 Oct. 1969), pp. 124-125, have shown that pore sizes of about 500 .ANG. are optimum for the separation of polystyrene molecules in the molecular weight range of 10,000 to 100,000 daltons.
It is known to use silica particles for adsorption of proteins such as interferon-gamma. For example, Pan et al., Eur. J. Biochem., 166: 145-149 (1987) discloses elution with 0.5M TMAC/TrisHCl, pH 8.0. Stankovic et al., Anal. Biochem., 184: 100-103 (1990) employs silica particles to elute gramicidin A with chloroform:methanol (1:1). Manning et al., J. Chromatog., 487: 41-50 (1989) reports use of derivatized silica for separating elastin peptides using methanol and iso-propanol. Skogen et al., Clin. Chem., 33: 401-404 (1987) discloses use of hydrophobic bonded-phase silica particles to extract digoxin-like immunoreactive factors, which are eluted with methanol/water. Byrne et al., Anal. Biochem., 148: 163-173 (1985) discloses isolation of gangliosides with consecutive chromatographies, including silica particles. Takagi et al., J. Chromatog., 208: 201-208 (1981) discloses the effect of salt concentration on the elution properties of complexes formed between sodium dodecyl sulphate and protein polypeptides in high-performance silica particle chromatography. Hillar et al., Bas. Appl. Histochem., 31: 299-313 (1987) uses a silica particle step with a methanol gradient in 0.1M ammonium acetate buffer on a C18 silica particle column to elute nuclear peptides from calf liver. See also Hillar et al., Physiol. Chem. Phys. Med. NMR, 17: 325-343 (1985) and Hillar et al., Physiol. Chem. Phys. Med. NMR, 17: 307-323 (1985), on purification using this solvent and butan-1-ol-propan-2-ol-acetic acid-water.
Mordarski et al., Archivum Immunologiae et Therapiae Exper., 25: 273 (1977) disclose purification of antibiotics on silica particles using 10% acetone in benzene and then methanol. Jentsch and Muecke, Int. J. Pept. Protein Res., 9: 78-79 (1977) disclose purifying bee venom peptides by chromatography on silica particles equilibrated with butanol-1-pyridine-acetic acid-water. Flouret et al., Int. J. Pept. Protein Res., 13: 137-141 (1979) purify oxytocin by adsorption chromatography on a silica particle column with a combination of methanol and chloroform. Stoffel et al., Hoppe-Seyler's Z. Physiol. Chem., 364: 1455-1466 (1983) purify proteolipid apoprotein by silica particle exclusion chromatography using 90% formic acid as solvent. Hemmasi et al., Hoppe-Seyler's Z. Physiol. Chem., 365: 485-492 (1984) discloses purification of peptide III (decosapeptide of bovine insulin B-chain) on silica particles and Sephadex LH-20 in chloroform: methanol ethylacetate/acetic acid. Trucksess and Stoloff, J. Assoc. Off. Anal. Chem., 62: 1080-1082 (1979) teaches purification of aflatoxin B1 and M 1 on a silica particle column, washing with chloroform, and eluting with chloroform-methanol.
See also Visser et al., Neth. Milk Dairy J., 29: 319-334 (1975); Loginova et al., Prikl. Biokhim. Mikrobiol., 14: 715-718 (1978); Schmidt et al., Anal. Chem., 52: 177-182 (1980); Lu et al., Shengwu Huazue Yu Shengwu Wuli Jinzhan, 48: 46-48 (1982); Wang et al., Gaodeng Xuexiao Huaxue Xuebao, 6: 557-561 (1985); Duhamel et al., Tetrahedron Lett., 26: 6065-6066 (1985); Pickart and Thaler, Prep. Biochem., 5: 397-412 (1975); Pillot and Petit, Mol. Immunol., 21: 53-60 (1984); Morise et al., Jpn Circ J., 52: 1309-1316 (1988); Wang et al., Biochemistry, 19: 5111-5117 (1980); Adachi et al., J. Chromatogr.. 428: 247-254 (1988); Singhal et al., Cancer Res., 47: 5566-5571 (1987); Tao et al., Biol. Chem. Hoppe Seyler, 368: 187-194 (1987); Mount et al., Arch. Biochem. Biophys., 240: 33-42 (1985); and Huennekens and Henderson, Chemistry and Biology of Pteridines, ed. by Pfleiderer (Berlin, de Gruyter, 1975).
A review of protein purification on porous glass is provided by Mizutani, J. Lig. Chromatog., 8: 925-983 (1985). On page 947, Mizutani states that addition of 5-10% ethanol in a buffer will increase the eluting force of a protein, with no supporting data or further information. Bock et al., Science, 191: 380-383 (1976) discloses protein purification by adsorption chromatography on controlled-pore glass using chaotropic buffers. Other references cited in the Mizutani review are MizuEani, J. Pharm. Sci., 69: 279-282 (1980) using 0.1% SDS during elution, Mizutani and Mizutani, J. Chrom., 168: 143-150 (1979) comparing elution of controlled-pore glass with CM-cellulose using standard proteins, Mizutani, J. Pharm. Sci., 69: 1226-1227 (1980) eluting antibodies from controlled-pore glass using 0.2M glycine (pH 8-9) and 0.1% SDS, and Yip et al., Proc. Natl. Acad. Sci. USA, 78: 1601-1605 (1981) eluting interferon-gamma with a combination of sodium chloride and ethylene glycol in phosphate buffer.
Additionally, in the U.S. patent literature, purification of proteins and other molecules using a silica particle absorbent is described by U.S. Pat. Nos. 4,908,432 issued Mar. 13, 1990; U.S. Pat. No. 5,057,426 issued Oct. 15, 1991; U.S. Pat. No. 4,777,242 issued Oct. 11, 1988 (adsorption of tumor necrosis factor onto silica glass beads, washing with buffer solution, and eluting with an aqueous lower alkanol, polyol, amine, or aminoalcohol at pH 8-11); U.S. Pat. No. 5,004,688 issued Apr. 2, 1991; U.S. Pat. No. 4,849,434 issued Jul. 18, 1989; U.S. Pat. No. 5,071,959 issued Dec. 10, 1991; U.S. Pat. No. 4,652,529 issued Mar. 24, 1987; U.S. Pat. No. 4,738,926 issued Apr. 19, 1988; U.S. Pat. No. 4,894,330 issued Jan. 16, 1990; U.S. Pat. No. 4,199,450 issued Apr. 22, 1980; U.S. Pat. No. 3,869,482 issued Mar. 4, 1975; U.S. Pat. No. 3,904,751 issued Sep. 9, 1975; U.S. Pat. No. 4,725,673 issued Feb. 16, 1988; and U.S. Pat. No. 3,876,775 issued Apr. 8, 1975.
Foreign published patent literature on the subject includes JP 1051097 published Feb. 27, 1989; JP 59159753 published Sep. 10, 1984; DD 209,187 published Apr. 25, 1984; EP 431,679 published Jun. 12, 1991; EP 337,492 published Oct. 18, 1989; DD 286,721 published Feb. 7, 1991; JP 90234692 published Sep. 17, 1990; DD 280,174 published Jun. 27, 1990; JP 51118810 published Oct. 19, 1976; JP 73008482 filed as JP 7063138 on Jul. 20, 1970; JP 50116691 published Sep. 12, 1975; JP 73019318 filed as JP 7033355 on Apr. 17, 1970; DD 217,823 published Jan. 23, 1985; DD 298,275 published Feb. 13, 1992; FR 2,653,034 published Apr. 19, 1991; SU 560,614 published Jul. 20, 1977; SU 1,272,227 published Nov. 23, 1986; CS 187,815 published Dec. 15, 1981; CS 243,336 published May 15, 1987; FR 2,600,341 issued Dec. 24, 1987; KR 9206401 published Aug. 6, 1992; WO 93/00361 published Jan. 7, 1993; and KR 9000748 published Feb. 15, 1990.
Chadha and Sulkowski, Prep. Biochem., supra, pioneered the use of tetramethyl ammonium chloride (TMAC) to elute proteins, specifically interferon-alpha, from controlled pore glass. They disclose that partial recovery of interferon-alpha was obtained with 50-75% ethylene glycol, 1M ammonium chloride, or 1M Tris-HCl. However, these reagents were not as selective as TMAC since they also eluted other proteins. See also Pan et al., supra. Even elution with TMAC has several disadvantages. For example, TMAC elutes several other proteins that are relatively hydrophobic compared to the protein insulin-like growth factor (IGF-I), which are difficult to remove from IGF-I in later steps.
IGF-I has been purified using gel filtration followed by ion-exchange on a sulfopropyl-substituted cation-exchange column, followed by buffer exchange and fractionation by a second gel filtration step. Next, preparative isoelectric focusing further separated the IGF-I from impurities with similar isoelectric points, followed by two reverse-phase chromatography steps to obtain pure IGF-I. Cornell et al., Prep. Biochem., 14:123 (1984). Clearly, because of the large number of steps involved, this protocol is relatively inefficient.
An alternative protocol for purifying IGF-I requires the fusion of Protein A to IGF-I by a linker, where the culture supernatant is passed through an affinity column consisting of IgG coupled to agarose. The IGF-I fusion product binds to the column while impurities pass through and the bound material is eluted, treated to remove the linker, and passed through IgG-agarose to remove the free Protein A. See Moks et al., Bio/Technology, 5: 379-382 (1987); Sofer, Bio/Technology, 4: 712-715 (1986).
IGF-I has been also purified by a series of adsorption-desorption steps employing a combination of cation-exchange and hydrophobic-interaction adsorbents. See U.S. Pat. No. 5,231,178 issued Jul. 27, 1993. Another method for purifying IGF-I involves centrifuging a human IGF-I-containing culture broth, diluting the supernatant with distilled water and adjusting the resulting solution to pH 5.6, passing the solution through a weak anion-exchange resin and 10 mM phosphate buffer solution (pH 5.6), dissolving the human IGF-I fraction in a 150-200 mM NaCl solution, and passing the fraction through a DE cation-exchange resin and 10 mM Tris buffer solution to obtain the final product. See KR 9208377 published Sep. 26, 1992. In addition, KR 9208378 published Sep. 26, 1992 discloses purifying IGF-I by passing a IGF-I-containing culture broth through an anion-exchange resin column, washing the column with phosphate buffer and dissolving with sodium-chloride-containing sodium carbonate solution to obtain the IGF-I fraction, passing the fraction through a cation-exchange resin column and concentrating, and passing the concentrated solution through a gel-filtration column to obtain the final product.
It is an object of the present invention to provide a general method of purifying polypeptides from hydrophobic impurities using only inexpensive underivatized silica particles.
It is another object of the present invention to provide a method of selectively eluting polypeptides from underivatized silica particles using a combination of ionic and organic solvents that does not destroy the silica particles.
It is a specific object of the invention to provide a method of purifying IGF-I from fermentation fluid.
These and other objects will become apparent to one of ordinary skill in the art.